Control of neural stem cells by the nascent cerebrospinal fluid
We propose to investigate how signals in the amniotic fluid (AF) and nascentcerebrospinal fluid (CSF) instruct neural stem cell behavior during early brain developmental stagessurrounding the time of neural tube closure (E8.5-E10.5). The rapidly changing and growing population ofneural stems at these early stages will give rise to all neurons and glia in the adult brain, yet comparatively fewstudies exist on the intrinsic genetic programs or the extrinsic fluid-based signals involved in driving these earlystages of development, largely due to technical limitations. We and others have demonstrated broad influencesof CSF and vascular fluid niches in instructing later stages of brain development. Yet virtually nothing is knownabout the protein composition of AF and early CSF during early brain development, at a time when the choroidplexus and vasculature have yet to form. Our overarching hypothesis is that secreted signals in the AF andnascent CSF are critical in instructing and synchronizing the proliferation and fate of embryonic E8.5-E10.5neural stem cells bathed in these fluids. We will first test this hypothesis by comparing the effects of AF, earlyCSF and buffered media on stem cell explants at ages E8.5 and E10.5. We predict that, as in laterdevelopment, early explants only develop normally when bathed in the age-appropriate fluid, suggesting thatchanges in the AF/CSF proteome are mirrored by changes in expression of associated receptors at the apicalsurface of neural stem cells. As such, we expect that normal stem cell function should be impaired by geneticperturbations to the cilia and membrane of neural stems cells at their apical surface, which is in direct contactwith the AF/CSF. A near-complete list of CSF proteins and their associated receptors on apical membranes willthen be deduced using quantitative mass-spec and RNAseq technologies, together with immunostaining. Wewill then determine the contributions of CSF-LIF and other top candidate proteins in instructing specific aspectsof E10.5 stem cell behavior, and the embryonic sources of these CSF signals. This proposal has importantclinical significance: We currently have relatively little understanding of the origins of early developmentaldisorders (defects due to errant neural tube closure, hydrocephalus, and infiltration by teratogens), and scantcapacities for early diagnosis or intervention. This proposal should provide a foundation for asking how theseearly perturbations ultimately derail some or all aspects of normal brain development, and for developing minimally invasive AF/CSF sampling and replacement strategies for disease diagnosis and for reprogramming of neural stem cells in order to bring the development brain back on track.